Collection, Direct Amplification, and Genotyping Methods for DNA Recovered from Pipe Bombs and Firearms

Improvised explosive devices (IEDs) and firearms have often been used to cause mass destruction and harm within communities. When a crime has been committed with these weapons, it is critical that a suspect or suspects be identified quickly. There are several approaches that can be used to identify a potential suspect, but few are as discriminatory as DNA analysis. Short tandem repeat (STR) analysis via capillary electrophoresis (CE) has proven to be robust method of human identification (HID); however, DNA remaining on touched or handled items is likely to be poor in quality and quantity, which can make STR profile interpretation extremely difficult or hinder it completely. Therefore, this study explores various collection strategies, novel genetic markers, and alternate analysis methods to increase the likelihood of retrieving usable genetic information from these challenging items. This was accomplished is four separate phases. The first study compared the informativeness of alternate genetic markers and analysis methods to standard STR analysis. PVC pipe bombs and copper wires were constructed and spiked with known quantities of biological material to recover mock low-template DNA from the surface PVC pipes and traces of blood from the ends of copper wires. DNA collected from PVC pipe fragments underwent traditional STR analysis as well as insertion/null (INNULs) analysis via CE and single nucleotide polymorphism (SNP) analysis via massively parallel sequencing (MPS) for HID. Blood from copper wires were analyzed using ancestry informative SNPs (AISNPs) via MPS. The results of this study showed that a complete INNUL profiles had a higher power of discrimination when less than 14 STR alleles were called. SNP analysis via MPS were the most discriminatory of all the methods tested when the same DNA concentration input was used. In addition, AISNPs correctly predicted the ancestry for five of the six blood samples tested. In the second study, three swab types and a swab storage device called the SwabSaver® were evaluated. Traditional cotton swabs, nylon-flocked swabs, and layered cotton paper swabs were used to collect DNA from handled assault rifle magazines and were then extracted or stored at room-temperature in a centrifuge tube or SwabSaver® device for one or two months. While nylon swabs resulted in higher DNA yields overall, swab storage had a greater impact on STR profile completeness, and swabs stored in the SwabSaver® device resulted in profiles comparable to swabs that were not stored. The third study examined two swab substrates and two direct amplification strategies for DNA collected from common pipe bomb substrates. PVC and steel pipes, electrical tape, and copper wires were spiked with known quantities of epithelial cells and swabbed with a conventional cotton swab or a microFLOQ® Direct swab (nylon-flocked swab). Swabs either underwent traditional DNA processing, direct amplification, or a pre-treatment strategy prior to STR amplification. The results of this proof-of-concept study showed that direct amplification using microFLOQ® Direct swabs was the most successful as it had the shortest processing time and resulted in the most complete STR profiles. Lastly, the fourth and final study examined two sub-sampling strategies for direct amplification using microFLOQ® Direct swabs. The first strategy involved DNA collection from firearms using a regular-tipped nylon swab, which was then sub-sampled using a microFLOQ® Direct swab. Rather than collecting from the larger swab head, the second strategy involved sub-sampling directly from post-blast pipe bomb substrates. A microFLOQ® Direct swab was used to collect from the fragments first and was followed with a more thorough collection with a regular-tipped nylon swab. All microFLOQ® Direct swabs underwent direct amplification while regular nylon swabs underwent traditional processing. Traditional DNA processing resulted in higher yields overall; however, sub-sampling from the larger swab head did result in partial and complete profiles. Sub-sampling from the pipe bomb substrates was mostly unsuccessful, which was likely due poor DNA deposition by the handler and high heat and pressure from the explosion